WO2023116639A1 - Procédé de préparation d'une puce à microsphères et application associée - Google Patents

Procédé de préparation d'une puce à microsphères et application associée Download PDF

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WO2023116639A1
WO2023116639A1 PCT/CN2022/140097 CN2022140097W WO2023116639A1 WO 2023116639 A1 WO2023116639 A1 WO 2023116639A1 CN 2022140097 W CN2022140097 W CN 2022140097W WO 2023116639 A1 WO2023116639 A1 WO 2023116639A1
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Prior art keywords
primer
microspheres
sequence
reaction
solution
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PCT/CN2022/140097
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English (en)
Chinese (zh)
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郑洪坤
刘敏
张梦龙
李佳
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北京百迈客生物科技有限公司
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Priority claimed from CN202111572805.XA external-priority patent/CN114350745B/zh
Priority claimed from CN202210137482.XA external-priority patent/CN114410764B/zh
Priority claimed from CN202220960413.4U external-priority patent/CN216712064U/zh
Priority claimed from CN202211139445.9A external-priority patent/CN115786457A/zh
Priority claimed from CN202211139855.3A external-priority patent/CN115725700A/zh
Priority claimed from CN202222884503.2U external-priority patent/CN218710327U/zh
Application filed by 北京百迈客生物科技有限公司 filed Critical 北京百迈客生物科技有限公司
Publication of WO2023116639A1 publication Critical patent/WO2023116639A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the field of biochip processing, in particular to a method for preparing a microsphere chip and related applications.
  • Biochip technology originated from the hybridization of nucleic acid molecules.
  • Biochips generally refer to micro-array hybrid chips (micro-arrays) of biological information molecules (such as gene fragments, DNA fragments or polypeptides, proteins) immobilized on mutual support media at high density.
  • the sequence and position of each molecule in the array are is known and is a preset sequence lattice.
  • Biochip technology is one of the most promising DNA analysis technologies at present, and the analysis objects can be nucleic acids, proteins, cells, tissues, etc.
  • the use of biochips for disease diagnosis is still in the research stage in the world. It has been used abroad to observe the expression and mutation of some genetic disease genes such as oncogenes and muscular atrophy.
  • Randomly adding coded microspheres on the microstructure processed chip is a biochip processing method, and this method has a low hole drop efficiency due to the error of the chip etching aperture and the inhomogeneity of the size of the microspheres. Certain upper limit. Usually, the porosity rate of microspheres is not high, and there are many residual microspheres on the chip.
  • the main purpose of the present invention is to provide a method for preparing a microsphere chip and its related applications, so as to solve the problems of complex biochip processing technology, low porosity rate and poor stability in the prior art.
  • the present invention provides the preparation method of microsphere chip (the method for silicon dioxide chip and microsphere self-assembly), comprising the following steps:
  • the diameter of micropores is similar to that of silica microspheres. It should be ensured that the size of the microspheres matches the micropores etched on the chip.
  • the main components of the UV-curable adhesive are base resin, active monomer and photoinitiator, such as Feifanli 3217 shadowless glue produced by Mizhan Technology Co., Ltd. It can also be purchased from Taobao manufacturers such as Kraft and Rongtai, preferably Feili.
  • the thickness of the film is roughly 1-3 microns (preferably 1 micron, thickness below 1 micron is not excluded here, such as 1nm, 10nm, 15nm, 20nm, 30nm, 50nm, 60nm, 70nm, 80nm or 90nm, etc.).
  • the thickness of the spin-coated UV adhesive should be as thin as possible and spin-coated evenly.
  • the depth of the microholes on the silica glass slide is 0.5-2.5 microns, preferably 1.5-2.5 microns (preferably about 2.1 microns, more preferably 1.5 microns, and the depth below 0.5 microns is not excluded here, to match the smaller microsphere size), the uniformly distributed microwells on the slide are calculated based on the total area of the slide of 7 mm ⁇ 7 mm, and the distance between the centers of two adjacent microwells is 4-6 microns (preferably about 5 microns, and 4 microns are not excluded here). The center point distance below micron, such as 2.5um, 3um, 3.5um, etc.).
  • step 3) centrifugation uses a plate centrifuge with a rotating speed of 1000rpm-3000rpm (preferably 2000rpm), and a centrifugation time of 10s-1min (preferably 30s).
  • the reagents used in step 3) to prepare the silica microsphere solution are ultrapure water, DMSO solution with a concentration of 5-20% by volume or an aqueous solution of UV-curable glue with a concentration of 2.5-10 ⁇ by volume.
  • the preferred concentration is 2.5-10 ⁇ of the UV-curable glue solution, more preferably 5 ⁇ of the UV-curable glue solution.
  • step 1) includes: immersing the silicon dioxide glass slide in the piranha solution for 30 minutes, then washing it with ultrapure water and absolute ethanol in sequence, and then drying it in the air.
  • the piranha solution is a mixture of concentrated sulfuric acid and hydrogen peroxide, wherein the volume ratio of concentrated sulfuric acid and hydrogen peroxide is 7:3.
  • the silica microspheres are covalently linked with nucleic acid, protein or polypeptide.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention is based on the method of treating silicon dioxide chips with ultraviolet curing glue (UV glue), and then assembling microspheres, which can achieve a porosity rate of more than 99% and less impurities.
  • UV glue ultraviolet curing glue
  • microsphere chip provided by the present invention is especially suitable for use as a spatial transcriptome chip.
  • the above-mentioned silica microspheres are silica microspheres covalently linked with long coding sequences, and further provide a method for preparing silica microspheres with long coding sequences, including the following step:
  • primer 1 Design four sets of primers, namely primer 1, primer 2, primer 3 and primer 4: the primers are all single-stranded oligonucleotides, primer 2 and primer 3 can anneal, and primer 1 and primer 4 The sequence length is the same;
  • the 15' end of the primer has an amino modification, and includes the READ1 sequence, barcode 1 sequence and linker 1 sequence of the Illumina sequencing platform from 5'-3';
  • the barcode 1 sequence is a coding sequence with a length of 10-20nt (including 12-768 species sequence), the linker 1 sequence is an auxiliary connection sequence of length 1-20nt;
  • Primer 2 is a coding sequence with a length of 10-20nt, i.e. barcode 2 sequence (including 12-768 sequences);
  • primer 3 The length of primer 3 is 12-60nt, including Linker 2 reverse complementary sequence, barcode 2 reverse complementary sequence and linker 1 reverse complementary sequence from 5′-3′;
  • Primer 4 includes linker 2 sequence, barcode 3 sequence (including 12-768 kinds of sequences), UMI sequence (random sequence) and polyT sequence from 5'-3'; Described barcode 3 sequence is the coding sequence of length 10-20nt, so The UMI sequence is a random primer with a length of 8-16nt (used to distinguish different transcripts), the length of the polyT sequence is 10-35nt, and the end contains a VN sequence; wherein V and N are degenerate bases, and V represents A and G or C, N means A, T, G or C;
  • primer 2 and primer 3 carry out annealing reaction
  • step (4) mixing the annealed product obtained in step (4) with the microspheres obtained in step (3) to carry out a DNA chain extension reaction;
  • step (6) Mix the microspheres obtained in step (5) with the primer 4, and perform a ligation reaction to obtain silica microspheres with long coding sequences.
  • primer 1 is the first-round primer
  • the annealed product formed by primers 2 and 3 is the second-round primer
  • primer 4 is the third-round primer
  • step (2) includes: mixing 4-10 mL of carboxylated silica microspheres with a concentration of 0.1-0.5 mg/mL after centrifugation and 4-10 mL of EDC and NHS mixed solution, and shaking the reaction at room temperature at 300-2000 rpm overnight.
  • EDC and NHS mixed solution is as follows: 10-30mg EDC and 5-30mg NHS are dissolved in 0.1-1M MES 1000-10000 ⁇ L to obtain the product.
  • step (3) includes: adding the activated microspheres into a 96-well plate, 10-40 ⁇ L per well, then adding 2-10 ⁇ L of primer 1 solution to each well, and shaking at 20-30° C. at 300-2000 rpm overnight ; After the reaction, wash the microspheres with PBS containing 0.001-0.03% v/v Tween-20, then wash the microspheres with TE buffer, and resuspend the washed microspheres with enzyme-free water.
  • the primer 1 solution is to dissolve the primer 1 in 0.1 M MES to obtain a primer 1 solution with a final concentration of 10-100 ⁇ M.
  • step (4) includes: mixing primer 2 and primer 3 in an equimolar ratio, adding 5 ⁇ annealing buffer to carry out annealing reaction; the annealing reaction conditions are: 95°C-15°C, annealing 1-10 °C, the annealed product was obtained.
  • the 5 ⁇ annealing buffer is: 10-50 ⁇ L of 1M Tris-HCl solution, 5-20 ⁇ L of 0.5M EDTA, 50-150 ⁇ L of 2M NaCl, and make up to 1000 ⁇ L with double-distilled water.
  • step (5) includes: adding the microspheres obtained in step (3) to a 96-well plate, 10-40 ⁇ L per well, and then adding 5 ⁇ L of 5 ⁇ T4 ligation buffer and 100-1000 U/ ⁇ L T4 ligase 2 to each well -10 ⁇ L, 20-100 ⁇ M annealed product 2-10 ⁇ L, make up to 50 ⁇ L with double-distilled water; shake and react at 16°C 300-2000rpm for 0.5-3h; then wash the microspheres with 5-20mM Tris-HCl solution of pH8, after washing Microspheres were resuspended in enzyme-free water.
  • step (6) includes: adding the microspheres obtained in step (5) to a 96-well plate, and then adding 5 ⁇ L of 5 ⁇ T4 ligation buffer, 100-1000 U/ ⁇ L T4 ligase 2-10 ⁇ L, and primer 4 solution to each well 2-10 ⁇ L, make up to 50 ⁇ L with double distilled water; shake and react at 16°C 300-2000rpm for 0.5-3h; then wash the microspheres with 10mM Tris-HCl solution of pH 8, and place the washed microspheres in 0.1-2M NaOH solution Melt in the medium, then wash the microspheres with enzyme-free water, and finally resuspend the microspheres with TE-TW solution, and store at 4°C.
  • the primer 4 solution is to dissolve the primer 4 in TE buffer solution with pH 8.0 to obtain a primer 4 solution with a final concentration of 10-100 ⁇ M.
  • TE-TW solution is TE buffer containing 0.01% Tween-20.
  • the T4 ligase used in the present invention can be purchased from Novozyme, Sangon, Yisheng, NEB and other companies.
  • silica microspheres prepared in the above preferred embodiments of the present invention can be used for further DNA decoding for spatial transcriptome sequencing.
  • microspheres prepared by this method are rich in types, using 12-768 kinds of barcode 1, barcode 2 and barcode 3, and finally carrying 1728 (12 ⁇ 12 ⁇ 12) or even 452984831 (768 ⁇ 768 ⁇ 768) different sequences
  • the microspheres can target more comprehensive tissues in spatial transcriptome sequencing, showing the transcriptome differences between different regions within the tissue.
  • the method provided by this preferred embodiment has high uniformity, and barcode 1, barcode 2 and barcode 3 of the present invention are more uniform, and the concentration of strict quantitative primers can effectively prevent a certain barcode from increasing or decreasing indefinitely.
  • the method provided by this preferred embodiment improves the reaction system of microsphere synthesis, makes the microsphere and the primer fully contact, and then connects by T4 ligase, so the connection efficiency of each step is higher, and the complete extension of the DNA sequence is guaranteed.
  • microspheres made of silica are used as marker carriers, and nano-silica microspheres are non-toxic, non-polluting, high-strength, high-toughness, good stability, and large surface area inorganic non-metallic Nanomaterials, so nanometer-scale monodisperse silica microspheres can be prepared on a certain scale, and have been widely used in biological cell separation and medical engineering.
  • Monodisperse silica microspheres have great application value in the synthesis of long-sequence-coded silica microspheres due to their good shape uniformity, controllable size, good dispersion properties, single composition, and easy surface functionalization.
  • the above-mentioned silica microspheres are silica microspheres covalently linked with long DNA sequences, and further provide a method for preparing the long DNA sequence silica microspheres, comprising the following steps:
  • primer 1, primer 2, primer 3 and UMI primer are all single-stranded oligonucleotides, and the sequence lengths of primer 1, primer 2 and primer 3 are the same, and the GC content Between 45-55%, ensure that the Tm value of each primer is similar;
  • Primer 1 (the first round of primers) has an amino modification at the 5 end, including the READ1 sequence, barcode 1 sequence and linker 1 sequence of the illumina sequencing platform from 5′-3′ (the other three rounds of primers synthesize the overall sequence by reverse complementary polymerization) ); primer 2 (the second round of primers) includes linker 2 reverse complementary sequence, barcode 2 reverse complementary sequence and linker1 reverse complementary sequence from 5'-3'; primer 3 (third round of primer) from 5'-3' 'include linker 3 reverse complementary sequence, barcode 2 reverse complementary sequence and linker2 reverse complementary sequence; UMI primer (fourth round primer) includes polyA sequence, UMI sequence (random sequence) and linker 3 reverse sequence from 5'-3' Complementary sequences; among them, barcode 1, barcode 2, and barcode 3 are different barcode sequences, and the length of polyA is 20-35nt;
  • Ligation reaction put the activated microspheres into the primer 1 solution, and perform condensation reaction to obtain silica microspheres with different sequences;
  • amino-modified oligonucleotide chain comprises READ1 sequence, barcode 1 and linker 1;
  • Step (2) includes: centrifuging 50 mg/mL carboxylated silica microspheres and mixing the precipitate with 20-100 ⁇ L of EDC and NHS mixed solution, shaking and reacting at room temperature at 1500-2000 rpm for 30 min-1 h.
  • the preparation method of EDC and NHS mixed solution is: dissolve 1.09mg EDC and 0.65mg NHS in 0.1M MES100ul to obtain.
  • Step (3) includes: mixing the activated microspheres with 2.5 ⁇ L of a 50 ⁇ M amino-modified oligonucleotide chain solution, and shaking and reacting at room temperature at 2000 rpm overnight; after the reaction is completed, the microspheres are collected by centrifugation, washed and used for subsequent synthesis reactions .
  • Washing of the microspheres included: placing the microspheres in 0.1M PBS containing 0.02% Tween 20, centrifuging to collect the microspheres, and then washing the microspheres twice with pH 8.0 TE buffer.
  • the polymerization reagent in step (4) comprises: dNTPs, Klenow enzyme and Klenow enzyme reaction buffer.
  • the reaction system used for DNA chain extension is: 50 ⁇ M primer 2 volume 1 ⁇ L, 10 ⁇ Klenow enzyme reaction buffer 5 ⁇ L, 2.5 mM dNTPs 4 ⁇ L, 5 U/ ⁇ L Klenow enzyme 1 ⁇ L.
  • the reaction conditions are: 37°C, 2000rpm shaking reaction for 30min-1h.
  • the reaction system used for chain extension of microspheres and UMI sequence in step (4) is: 50 ⁇ M UMI primer volume 1 ⁇ L, 10 ⁇ Klenow enzyme reaction buffer 5 ⁇ L, 2.5mM dNTPs 4 ⁇ L and 5U/ ⁇ L Klenow enzyme 1 ⁇ L.
  • the reaction conditions are: 37°C, 2000rpm shaking reaction for 0.5-1h.
  • the nucleotide sequence of primer 1 is shown in SEQ ID NO: 1-5
  • the nucleotide sequence of primer 2 is shown in SEQ ID NO: 6-10
  • the nucleotide sequence of primer 3 is shown in SEQ ID NO Shown in: 11-15
  • the nucleotide sequence of UMI primer is shown in SEQ ID NO: 16.
  • the carboxylated silica microspheres used in the long DNA sequence silica microspheres are purchased from Shanghai Carboxyphene Biomedical Technology Co., Ltd., and can also be prepared according to conventional methods.
  • This preferred embodiment provides a method for synthesizing long-sequence-coded carboxylated silica.
  • the method uses silica as a carrier and uses chemical reagents to activate surface carboxyl functional groups, which can be combined with amino-modified oligonucleotide sequences. valence connection.
  • subsequent sequences can be introduced (sequence extension) by means of annealing of complementary sequences combined with polymerization.
  • a variety of long-sequence-coded silica microspheres can be obtained. Compared with the traditional method, this method has the advantage that there are more oligonucleotide sequences modified on the silica microspheres, and the biological information contained is more comprehensive.
  • EDC and NHS can fully activate the carboxyl structure modified on the surface of silica microspheres.
  • a spatial transcriptome chip is also provided, which is used to solve the defects of low resolution and high cost of sample analysis existing in biochips in the prior art. Analysis resolution.
  • the spatial transcriptome chip provided by the present invention includes:
  • a substrate forms a plurality of rectangular microporous regions, and forms a plurality of sub-rectangular microporous regions inside the rectangular microporous region, and each of the sub-rectangular microporous regions is uniformly distributed with a plurality of microporous structures, so The above-mentioned microporous structure is used to place the coded microspheres.
  • gaps are formed between adjacent sub-rectangular microwell regions, and the size of each gap is equal.
  • the width of the gap ranges from 2 microns to 20 microns.
  • At least one of the sub-rectangular microwell regions located on the four right angles of the rectangular microwell region is provided with a label.
  • gaps are formed in the sub-rectangular microwell area located at any three positions of the four right angles of the rectangular microwell area to form markers.
  • the notch is located at any right-angle position of the sub-rectangular microwell area.
  • the diameter of the micropore structure ranges from 1 micron to 10 microns.
  • the length and width ranges of the rectangular microwell area are both between 5 mm and 20 mm.
  • the value ranges of the length and the width of the sub-rectangular microwell area are both between 100 microns and 300 microns.
  • the substrate is a glass substrate, and the glass substrate forms a microporous structure by etching.
  • a spatial transcriptome chip provided by the present invention is constructed with a plurality of rectangular microwell areas on the substrate, and a plurality of sub-rectangular microwell areas are also formed in each rectangular microwell area, and each sub-rectangular microwell area is evenly distributed with
  • the microporous structure improves the resolution of the spatial transcriptome through the microporous structure, realizes high-sensitivity detection, and meets the needs of scientists for the analysis of subcellular structures; and the glass substrate can be used to make the microporous structure by etching technology, and the manufacturing process is simple , Reduce the cost of consumables.
  • a spatial transcriptome biochip is also provided, which is used to solve the problem that the non-transparent substrate of the biochip in the prior art cannot be used for HE staining, toluidine blue staining and Masson's staining.
  • the shortcomings of bright-field microscopic imaging such as staining and the use of slide substrates cannot meet the needs of scientists for subcellular structure analysis.
  • a spatial transcriptome biochip provided by some preferred embodiments, including:
  • a transparent substrate forms a microporous area by photolithography and etching, and a microporous structure is formed in the microporous area for placing coded microspheres with primers; the microporous area forms at least one sub-area, The sub-area is a circular area or a polygonal area.
  • a plurality of micro-units are formed in the sub-region, adjacent micro-units are arranged at intervals, and the micro-units are arranged repeatedly in circles or polygons.
  • notches are formed on the edges and/or corners of the tiny units to form orientation marks.
  • the distance between the micro-units is between 0 microns and 40 microns.
  • the diameter of the micropore structure ranges from 0.1 microns to 10 microns, and the center point distance between two adjacent microwells is from 0.1 microns to 20 microns.
  • the long side of the transparent substrate has a value range of 10-100 mm, and the short side has a value range of 5-50 mm.
  • the size of the sub-region ranges from 9 mm 2 to 1875 mm 2 .
  • the transparent substrate is one of glass, quartz, plastic, magnesium chloride and gallium arsenide.
  • the microporous structure includes a flaring portion located on the surface of the transparent substrate and a constricting portion located inside the transparent substrate, the flaring portion and the The constriction communicates along the depth direction of the transparent substrate to form the microporous structure on the transparent substrate.
  • the transparent substrate has a first surface and a second surface, and the first surface and the second surface respectively form a micropore area by photolithography and etching; wherein , the first surface and the second surface are two opposite surfaces of the transparent substrate.
  • the spatial transcriptome biochip uses a transparent substrate, uses photolithography and etching techniques to make a microporous structure and forms a microporous region, and has a simple manufacturing process and reduces the cost of consumables, and Bright field imaging can be combined with gene expression results, which greatly improves the analysis effect of spatial transcriptome; in addition, the micropore structure of this chip has high resolution, which can realize the analysis of spatial transcriptome data at subcellular level.
  • a three-color fluorescence decoding method based on a subcellular level spatial chip comprising the following steps:
  • Hybridization between the chip and the decoding probe mix the decoding probe I, decoding probe II and decoding probe III with different fluorescent labels, and then perform a hybridization reaction with the biochip, and then detect the corresponding fluorescent signal;
  • Chip unzipping and cleaning Melt the hybridized biochip with sodium hydroxide, wash and dry the unzipped biochip, and hybridize the next round of decoding probes again;
  • primer 1, primer 2 and primer 3 each include 4-384 kinds of primer sequences; primer 1, primer 2 and primer 3 are sequentially connected in series according to 5'-3';
  • Decoding probe I is a single-stranded oligonucleotide complementary to the barcode of primer 1
  • decoding probe II is a single-stranded oligonucleotide complementary to the barcode of primer 2
  • decoding probe III is complementary to the barcode of primer 3
  • Oligonucleotides are single-stranded.
  • the silica microspheres described in step A are carboxylated silica microspheres.
  • step A includes: connecting 4-384 kinds of primer sequences of primer 1 to carboxyl silica microspheres through aminocarboxyl condensation reaction, and after the connection is completed, the microspheres are mixed and cleaned, and evenly divided into 4-384 parts, each Add 4-384 kinds of primer sequences of primer 2 for the ligation reaction. After the connection is completed, the microspheres are mixed and washed, and evenly divided into 4-384 parts. Each part is added with 4-384 kinds of primer sequences of primer 3 for the ligation reaction. After the connection is completed , spread the microspheres evenly on the microporous glass plate, and fix them at the hole positions to obtain the biochip.
  • the fluorescent label used in the present invention can be selected from DAPI, FITC, Alexa fluor 488, Cy2, Cy3, Cy5, Cy5.5, TRITC, Cy7, etc.
  • the hybridization reaction described in step B is carried out in a hybridization buffer; the composition of the hybridization buffer is: 1-10mM NaCl, 2-5mM Tris-HCl, 1-3mM MgCl and 0.5-5mM DTT (disulfide threitol).
  • step B the concentration of the decoded probe after mixing is 1nM-50nM, and 10 ⁇ L of the mixed decoded probe is mixed with 40-190 ⁇ L of hybridization buffer, added to the wells, and hybridized with the biochip.
  • the conditions for the hybridization reaction in Step B are: 37-60° C. for 5-20 minutes.
  • step C includes:
  • step C12 Repeat step C12 to 3 times to ensure complete unzipping
  • the unzipped biochip is washed with enzyme-free water for 1 to 3 times, dried and hybridized with the next round of decoding probes again.
  • this preferred embodiment provides the application of the method in the study of subcellular spatial omics (especially the study of spatial location information of tissues at the subcellular level).
  • microspheres can be combined in a variety of ways, so there are many sequences that can be used for decoding.
  • Fig. 1 is a schematic flow chart of the silicon dioxide chip and microsphere assembly method of the present invention.
  • Fig. 2 is an effect diagram of a silicon dioxide chip before assembly in a preferred embodiment of the present invention.
  • Fig. 3 is an effect diagram after self-assembly of silicon dioxide chips and microspheres in a preferred embodiment of the present invention.
  • FIG. 4 is an effect diagram of a silicon dioxide chip after being treated at 95° C. for 30 minutes in a preferred embodiment of the present invention.
  • Fig. 5 is a schematic diagram of the sequence structure of silica microspheres synthesized by the ligation method of the present invention to encode long sequences.
  • Fig. 6 is a fluorescent quality inspection picture of silica microspheres synthesized by the ligation method of the present invention with long sequence coding.
  • Fig. 7 is a fluorescent quality inspection image of silica microspheres encoded by a long sequence synthesized in CN114410764A.
  • Fig. 8 is a schematic diagram of the structure of the long-sequence-coded silica microspheres synthesized in Example 7 of the present invention.
  • Fig. 9 is a schematic structural diagram of a spatial transcriptome chip provided in a preferred embodiment of the present invention.
  • Fig. 10 is a schematic diagram of the structure of the rectangular microwell area in the spatial transcriptome chip provided in a preferred embodiment of the present invention.
  • Fig. 11 is a schematic diagram of the structure of the sub-rectangular microwell area in the spatial transcriptome chip provided in a preferred embodiment of the present invention.
  • Fig. 12 is a schematic structural view of the sub-rectangular micropore region forming the first type of notch provided in a preferred embodiment of the present invention.
  • Fig. 13 is a schematic structural view of the sub-rectangular micropore region forming the second type of gap provided in a preferred embodiment of the present invention.
  • Fig. 14 is a schematic structural diagram of the sub-rectangular micropore region forming a third type of notch provided in a preferred embodiment of the present invention.
  • 101 substrate; 201: rectangular microporous area; 301: sub-rectangular microporous area; 70: microporous structure; 50: notch; 60: gap.
  • Fig. 15 is a schematic diagram of the sub-region in a large rectangular shape in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 16 is a schematic diagram of the sub-region in the shape of a small rectangle in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 17 is a schematic diagram of the structure of the circular sub-region in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 18 is a schematic diagram of the hexagonal sub-region in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 19 is a schematic diagram of the structure of micro-units arranged uniformly in a rectangle in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 20 is a schematic diagram of the structure of micro-units arranged uniformly in hexagons in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 21 is a schematic diagram of the structure of the space transcriptome biochip provided in a preferred embodiment of the present invention where the notch is located at the edge of the tiny unit.
  • Fig. 22 is a schematic diagram of the structure of the notch located at the corner of the tiny unit in the spatial transcriptome biochip provided in a preferred embodiment of the present invention.
  • Fig. 23 is a schematic diagram of the structure of the spatial transcriptome biochip provided in a preferred embodiment of the present invention without gaps formed in micro-units.
  • Fig. 24 is a longitudinal cross-sectional view of a spatial transcriptome chip provided in a preferred embodiment of the present invention.
  • 10 transparent substrate; 11: first surface; 12: second surface; 20: microporous region; 30: subregion; 40: microunit; 50: gap; 60: gap; 70: microporous structure; 71: expanded Mouth; 72:shrinking mouth.
  • Figure 25 shows that after the microspheres are hybridized with different decoding probes, the corresponding sequence structure is obtained according to the color arrangement of the fluorescent labels in each round, thereby realizing the chip decoding process.
  • a method for assembling a silicon dioxide chip and microspheres is provided.
  • a silicon dioxide glass slide etched with micropores is used as a substrate, and a layer of ultraviolet glue is evenly applied on it ( After ultraviolet light curing glue), coded silica microspheres (that is, silica microspheres covalently linked with nucleic acid molecules) are added, and after centrifugation, a biochip with spatial decoding ability is obtained.
  • the present invention provides a simple and low-cost way to realize the rapid fabrication of the spatial transcriptome chip.
  • the silica chip is cleaned first, then the surface microspheres are coated with UV glue, and then the coded free silica microspheres are added, and the microspheres fall onto the silica chip by centrifugation through a spin coating device, and finally after Dry and clean to get a microarray suitable for spatial transcriptome experiments. Chips can be scanned with a microscope to evaluate the dropout effect.
  • Chip coating treatment use UV glue to smear the target area of the chip, and use it for later use after ultraviolet irradiation;
  • Microsphere filling Dilute the coded microspheres with ultrapure water, then evenly spread the microspheres on the target area of the chip, let stand for 4 minutes, and then use a spin coating device (plate centrifuge) to centrifuge;
  • Chip cleaning remove the residual suspension above the target area, and clean it with a brush after drying;
  • Chip quality control put the assembled spatial transcriptome chip under a scanning microscope to take pictures and scan.
  • the UV glue used in the following examples is Feifanli 3217 shadowless glue produced by Mizhan Technology Co., Ltd.
  • the plate centrifuge was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., model OSE-MP25.
  • the diameter of the silica microspheres is 2.5 microns
  • the depth of the microwells on the silica chip is 2.1 microns
  • the microwells evenly distributed on the slide are calculated according to the total area of the slide 7mm ⁇ 7mm, the distance between the centers of two adjacent microwells The distance is 5 microns.
  • the cells and tissues of higher organisms have high spatial heterogeneity.
  • the relative position of cells in tissue samples and the spatial information of gene expression are very important for the study of disease pathology and biological development.
  • the resolution and detection throughput of single-cell sequencing technology have been greatly improved, enabling researchers to obtain the heterogeneity between cells at single-cell resolution.
  • spatial transcriptome a spatially barcoded RNA-Seq approach, provides researchers with spatial information about where cells are located in a tissue, as well as the cellular composition and gene expression status of different regions in the tissue, but existing methods require pre-selection of markers, Moreover, the latest spatial transcriptome technology does not yet reach the resolution of single cells, so it is more common to combine spatial transcriptome with single-cell sequencing technology.
  • the first step of spatial transcriptome sequencing can be through the preparation of markers with barcodes, fixed markers on biochips, or random fixation followed by marker decoding.
  • a method for synthesizing silica microspheres encoded by long sequences by ligation is also provided.
  • carboxyl-modified silica microspheres are used as carriers, which are covalently linked with amino-modified oligonucleotide sequences.
  • T4 ligase catalyzes the phosphodiester bond between the 5'-P end and the 3'-OH end of the subsequent oligonucleotide sequence.
  • This method can obtain a variety of barcode silica microspheres. Compared with the traditional PCR method to synthesize Barcode microspheres, the method has the advantages of reducing the deviation of the reaction, improving the connection efficiency, and the operation is simple and easy, and the application prospect is broad.
  • the present invention provides a new long-sequence-coded silica microsphere synthesis method.
  • the carboxyl sites on the surface of the silica microspheres are activated to expose the carboxyl groups on the surface, and then combined with 12, 24, 48, 96, Condensation reaction of 192, 288, 384, 768 or more oligonucleotides with unique Barcode 1 sequences; further catalyzed by T4 ligase between the 5'-P end and the 3'-OH end of the oligonucleotide Form phosphodiester bonds, introduce 12, 24, 48, 96, 192, 288, 384, 768 and even more Barcode 2 sequences; finally use T4 ligase again to introduce 12, 24, 48, 96, 192, 288, 384, 768 Barcode 3 sequences.
  • the present invention adopts following technical scheme:
  • primers a total of 4 sets of primers, namely primer 1, primer 2, primer 3 and primer 4.
  • the primers are all single-stranded oligonucleotides, wherein primer 2 and primer 3 are complementary sequences, annealed to be double-stranded for use; primer 1 and primer 4 have the same sequence length;
  • the first round of primers Primer 1, which has an amino modification at its 5' end, and is covalently bonded to the carboxyl modification site on the surface of the silica microsphere. From 5′-3′, it includes the READ1 sequence, Barcode 1 sequence, and Linker 1 sequence of the Illumina sequencing platform.
  • the Barcode 1 sequence is a 10-20nt coding sequence and Linker 1 is a 1-20nt auxiliary connection sequence;
  • the second round of primers : Including primer 2 and primer 3, primer 2 is a 10-20nt oligonucleotide single strand for coding, that is, the Barcode2 sequence.
  • Primer 3 is a 12-60nt oligonucleotide single strand, including the complementary pairing sequence of primer 2, the auxiliary connection sequence complementary to Linker 1 and Linker 2; the third round of primers: primer 4, including from 5'-3' Link 2 sequence, Barcode 3 sequence, UMI sequence (random sequence) and PolyT sequence, where Linker 2 is an auxiliary connection sequence with a length of 1-20nt, Barcode 3 sequence is a 10-20nt coding sequence, and UMI is a 8-16nt Random primers are used to distinguish different transcripts, the length of PolyT is 10-35nt, and the end contains VN sequence (degenerate base).
  • primer 4 including from 5'-3' Link 2 sequence, Barcode 3 sequence, UMI sequence (random sequence) and PolyT sequence, where Linker 2 is an auxiliary connection sequence with a length of 1-20nt, Barcode 3 sequence is a 10-20nt coding sequence, and UMI is a 8-16nt Random primers are used to distinguish different transcripts, the length of
  • step (2) add amino-modified Barcode 1 oligonucleotides (including READ1 sequence, Barcode 1 sequence and Linker 1 sequence), and shake the metal bath for 1h, 2h, 4h, 8h , 12h, 24h and other times.
  • amino-modified Barcode 1 oligonucleotides including READ1 sequence, Barcode 1 sequence and Linker 1 sequence
  • Barcode 2, 3 ligation reaction Add the annealing primer in (4) to the microspheres in (3) for DNA chain extension reaction; the obtained microspheres are then added to the Barcode 3 oligonucleotide column (comprising Linker 2 sequence, Barcode 3 sequence, UMI sequence and PolyT sequence) for further ligation reactions, and finally obtained 12 ⁇ 12 ⁇ 12 to 768 ⁇ 768 ⁇ 768 long-sequence-coded silica microspheres.
  • FIG. 5 The schematic diagram of the sequence structure of silica microspheres with long sequence codes synthesized by ligation method is shown in Fig. 5 .
  • nucleic acid including deoxyribonucleic acid and ribonucleic acid
  • nucleic acid molecules are closely related to and play an important role in the occurrence and development of various diseases that affect human health. Therefore, the development of accurate and effective methods for sensitive and accurate detection is of great significance for in-depth exploration of nucleic acid functional regulation, drug screening, early detection, clinical diagnosis and treatment, and prognosis evaluation of related diseases.
  • Silica is a typical inorganic powder material with large specific surface area and good chemical stability. Monodisperse silica microspheres have a simple preparation process and good biocompatibility, so silica microspheres with different sizes and surface modifications can be used in different fields such as information, biology, and medicine.
  • nucleic acid contains a lot of biological information and a large amount of data. Therefore, the oligonucleotide chain with long sequence coding is modified on the substrate, and then used for DNA sequencing. The collection of information is of great significance.
  • a method for synthesizing silica microspheres encoded by long DNA sequences is also provided.
  • This method adopts carboxylation reaction to connect carboxylated silica and the sequence with amino group, then adds the complementary sequence of subsequent barcode sequence, and uses primer annealing and polymerase polymerization reaction to obtain silica with long sequence coding Microspheres, so that the obtained microspheres are further applied to DNA chip decoding.
  • Design primers design coding primer sequences.
  • Activation of carboxylated silica microspheres use EDC and NHS to activate the silica microspheres with carboxyl groups to obtain silica microspheres with good dispersion and exposed carboxyl groups on the surface.
  • Ligation reaction Carboxylated silica microspheres and oligonucleotide chains with amino-terminal modification are connected through carboxylation reaction to obtain silica microspheres with different sequences.
  • Gene expression patterns at spatially native locations in tissues are important for understanding the types and functions of cells within them.
  • spatial transcriptome technology has developed rapidly and is widely used in different fields such as tumors, diseases, nervous system and organ development.
  • the carriers currently used to make biochips generally have active groups that can undergo chemical reactions, so as to be used for coupling biomolecules.
  • the types of such carriers mainly include glass slides, silicon wafers, nitrocellulose membranes, nylon membranes, etc.
  • the chip of the spatial transcriptome in the prior art uses a glass slide, which is a durable carrier that can withstand high temperature and high ionic strength, and has non-wetting properties, which minimizes the hybridization volume; in addition, the hydrophobic surface overcomes the The disadvantage that the sample is easy to diffuse increases the density of the sample point; the low fluorescence signal will not cause strong background interference.
  • the biochip immobilizes the capture probes for spatial analysis on glass slides, but it has the defect of low resolution of sample analysis. Therefore, how to improve the resolution of sample analysis and reduce the cost of consumables, so as to meet the needs of scientists for subcellular structures The need for analysis is an urgent problem to be solved.
  • the spatial transcriptome chip includes: a substrate 101; wherein, the substrate 101 forms a plurality of rectangular microwell areas 201, and forms a plurality of sub-rectangular microwell areas 301 inside the rectangular microwell area 201, and each sub-rectangular microwell area 301 is uniformly distributed There are a plurality of microporous structures 70, and the microporous structures 70 are used to place the encoded microspheres.
  • the substrate 101 is a glass substrate, and the glass substrate is etched to form microporous structures 70, and the microporous structures 70 are all round holes.
  • the uniformly distributed microporous structure 70 forms a sub-rectangular microporous area 301 , a plurality of sub-rectangular microporous areas 301 constitute a rectangular microporous area 201 , and a plurality of such rectangular microporous areas 201 are constructed on a glass substrate.
  • the length value range and the width value range of the rectangular micropore area 201 are both between 5 mm and 20 mm; between 300 microns; the diameter of the microporous structure 70 ranges from 1 micron to 10 microns, and the minimum distance between the centers of adjacent microporous structures 70 ranges from 1 micron to 2 microns.
  • this preferred embodiment provides a size design of a spatial transcriptome chip
  • the length, width and thickness of the glass substrate are 75 mm, 25 mm and 1 mm respectively
  • eight rectangular microwell regions 201 are arranged on the glass substrate, And set four rows along the length direction, set two columns along the width direction, the distance between the rectangular microporous area 201 and the top of the glass substrate is 8mm, and the distance from the side of the glass substrate is 3.3mm, between the two columns of rectangular microporous areas 201 4 mm apart, and 5 mm apart the rectangular microwell areas 201 preceding adjacent rows.
  • the length and width of each rectangular microporous area 201 are 7.2 mm and 7.2 mm, respectively.
  • the pore diameter of the microporous structure 70 etched on the substrate 101 is about 2.5um, which is equivalent to increasing the resolution of the current mainstream spatial transcriptome by more than 20 times.
  • HE staining and gene expression experiments can also be performed on glass slides later.
  • a spatial transcriptome chip provided in this embodiment is constructed with a plurality of rectangular microwell regions 201 on a substrate 101, and a plurality of sub-rectangular microwell regions 301 are also formed in each rectangular microwell region 201, each sub-rectangular microwell region Microporous structures 70 are evenly distributed in the area 301 (as shown in FIG. 11 ), through which microporous structures 70 can improve the resolution of the spatial transcriptome, realize high-sensitivity detection, and meet the needs of scientists for subcellular structure analysis; and glass can be used
  • the microporous structure 70 is fabricated by etching technology, the fabrication process is simple, and the cost of consumables is reduced.
  • gaps 60 are formed between adjacent sub-rectangular microwell regions 301 , and the size of each gap 60 is equal. Specifically, the width of the gap 60 ranges from 2 microns to 20 microns. It should be noted that the width of the gap refers to the distance between the centers of the two microporous structures 70 .
  • the scanning device scans the microporous structure 70 on the substrate 101, it is necessary to use a software algorithm to stitch the scanned images. There are a large number of 70, and splicing errors often occur during the splicing process.
  • the width of the gap 60 can be processed according to a specific scanning device and software algorithm, and the present invention is not limited to the value range of the width of the gap 60 mentioned above.
  • At least one sub-rectangular microwell area 301 in the sub-rectangular microwell area 301 located on the four right angles of the rectangular microwell area 201 is provided with a label .
  • a mark is set in the sub-rectangular micro-hole area 301 at a right angle to the rectangular micro-hole area 201, so that it is convenient to distinguish the orientation and identify the rotation of the image.
  • the direction and angle are more conducive to improving the accuracy of image processing. It can be understood that the sub-rectangular micropore regions 301 in different rectangular micropore regions 201 can be marked in different forms to facilitate identification.
  • the sub-rectangular micro-hole area 301 located at any three positions in the four right angles of the rectangular micro-hole area 201 forms a gap 50 to form a mark; while the sub-rectangular micro-hole area 301 on the other right angle has no gap 50, is a normal rectangular area.
  • the notch 50 is located at any right-angle position of the sub-rectangular microhole area 301 .
  • the shape of the notch 50 may be a rectangle as shown in FIGS. 12 and 13 , or a triangle as shown in FIG. 14 .
  • a notch is etched at a right angle position of the rectangular microhole area 301 to form a mark, which is convenient for distinguishing the orientation and identifying the rotation direction and angle of the image.
  • biochips As previously mentioned, gene expression patterns at spatially native locations in tissues are important for understanding cell types and functions within them.
  • spatial transcriptome technology has developed rapidly and is widely used in different fields such as tumors, diseases, nervous system and organ development.
  • the carriers currently used to make biochips generally have active groups that can undergo chemical reactions, so as to be used for coupling biomolecules.
  • the types of such carriers mainly include glass slides, silicon wafers, nitrocellulose membranes, nylon membranes, etc.
  • this durable carrier can withstand high temperature and high ionic strength, and has non-wetting properties, which minimizes the volume of hybridization; in addition, the hydrophobic surface overcomes the shortcomings of easy diffusion of samples and improves the quality of samples. Dot density; low fluorescence signal does not cause strong background interference.
  • the biochip immobilizes the capture probes for spatial analysis on glass slides, but it has the defect of low sample analysis resolution, which cannot meet the needs of scientists for subcellular structure analysis.
  • the chip of this material is developed based on gene sequencing, which can realize the analysis of the spatial transcriptome at the subcellular level, but the substrate of non-transparent material cannot be used for HE staining, toluidine Bright-field microscopic imaging such as blue staining and Masson staining can only realize tissue imaging by means of fluorescence, or use clinical slides for bright-field imaging, resulting in that the final gene expression results cannot be well combined with the microscope collection results.
  • a spatial transcriptome biochip with improved structure is provided.
  • a spatial transcriptome biochip of this preferred embodiment is described below with reference to FIGS. 15-24 , including: a transparent substrate 10 .
  • the transparent substrate 10 forms a microporous area 20 by photolithography and etching, and a microporous structure 70 is formed in the microporous area 20, and the microporous structure 70 is used to place the coded microspheres with primers.
  • the transparent substrate 10 in this embodiment can be made of glass, quartz, plastic or transparent conductive coating; the transparent conductive coating can be one of magnesium chloride, gallium arsenide and the like.
  • the microporous region 20 is formed on the transparent substrate 10 by means of photolithography and etching, and the microporous structure 70 in the microporous region 20 can place the coded microspheres with primers.
  • the microporous region 20 in this embodiment can be a microporous region 20 formed by uniformly paving the microporous structure 70 as a whole, and can also form subregions 30 of different shapes (specific examples are given in the following embodiments), according to different shapes
  • the sub-region 30 can be more beneficial for different scanning software to splicing and recognizing images.
  • the microporous structure 70 improves the resolution of the spatial transcriptome, realizes high-sensitivity detection, and meets the needs of scientists for the analysis of subcellular structures; and the glass substrate can be used to manufacture the microporous structure 70 by etching technology, and its manufacturing process is simple, reducing Supplies cost.
  • the spatial transcriptome biochip in this example can perform bright-field microscopic imaging such as HE staining, toluidine blue staining, and Masson staining, and can combine gene expression results with bright-field staining results for comparative analysis.
  • the spatial transcriptome biochip provided by this preferred embodiment adopts a transparent substrate, uses photolithography and etching technology to make a microporous structure and forms a microporous area, and its manufacturing process is simple, reducing the cost of consumables, and can Field imaging, combined with gene expression results, greatly improves the analysis effect of spatial transcriptome; in addition, the micropore structure of this chip has high resolution, which can realize the analysis of spatial transcriptome data at subcellular level.
  • the microporous area 20 forms at least one sub-area 30, and the sub-area 30 is a circular area or a polygonal area.
  • the subregion 30 in this embodiment is also composed of a plurality of microporous structures 70 , and the plurality of subregions 30 form an entire microporous region 20 according to a specific arrangement.
  • the sub-region 30 is a large rectangle, as shown in Figure 16, the sub-region 30 is arranged in multiple rows and rows of small rectangles, and as shown in Figure 17, the sub-region 30 is arranged in two circles, as shown in Figure 17.
  • the sub-regions 30 shown in 18 are arranged in four hexagons.
  • the sub-region 30 can be one as shown in FIG. 1 , or multiple as shown in FIG. 16 , FIG. 17 and FIG. 18 , and the shape of the sub-region 30 can be not only a circle, but also a triangle. , rectangle, hexagon and other polygons, examples are not given here.
  • a plurality of tiny units 40 are formed in the sub-region 30 , adjacent micro units 40 are arranged at intervals, and the micro units 40 are arranged evenly or repeatedly in a circle or polygon.
  • a plurality of smaller micro-units 40 are also formed in the sub-region 30, and the micro-units 40 in the same sub-region 30 are evenly arranged in a circular or polygonal shape, such as the micro-units 40 shown in FIG. 19 They may be evenly arranged in a rectangle, or may be arranged in a hexagon as shown in FIG. 20 . It can be understood that, as shown in FIG.
  • the tiny unit 40 is also composed of a plurality of microporous structures 70 , and the plurality of tiny units 40 form a sub-region 30 according to a specific arrangement.
  • the scanning device scans the microporous structure 70 on the substrate, it is necessary to use a software algorithm to stitch the scanned images. There are a large number of them, and splicing errors often occur during the splicing process.
  • the sub-region 30 is divided into a plurality of tiny units 40 with smaller sizes, which is convenient for scanning equipment and software to identify images in different regions, and facilitates later stages. The correction of image acquisition avoids the positioning error of chip coding information, thereby improving the splicing accuracy.
  • the size range of the sub-region 30 is between 9 mm 2 and 1875 mm 2 , of course, according to different shapes of the sub-region 30 and different experimental needs, its size can also be adjusted accordingly.
  • the distance between the tiny units 40 is between 0 ⁇ m and 40 ⁇ m, and the same applies to the tiny units 40 .
  • notches 50 are formed on the edges and/or corners of the tiny units 40 to form orientation marks.
  • orientation marks are set on the edge and/or angular position of the tiny unit 40 located at the edge and/or angular position of the sub-region 30, so as to facilitate Distinguishing the orientation and identifying the rotation direction and angle of the image is more conducive to improving the accuracy of image processing.
  • the tiny unit 40 provided with the gap 50 in this embodiment refers to the tiny unit 40 located at the edge and/or corner of the sub-region 30, the shape of the gap 50 can be a rectangle, a triangle, etc., the gap 50 A part of the tiny unit 40 is cut out to form an orientation mark, which is convenient for distinguishing the orientation and identifying the rotation direction and angle of the image. And because the shape of the tiny unit 40 can be circular or polygonal, thus, the notch 50 can be positioned at the edge of the tiny unit 40 as shown in Figure 7; The position of the corner; FIG. 23 is a schematic diagram of not forming a gap 50 in the micro unit 40 .
  • the diameter of the microporous structure 70 ranges from 0.1 microns to 10 microns, and the distance between the center points of two adjacent micropores ranges from 0.1 microns to 20 microns.
  • the microporous structure 70 is formed inside, and the microporous structure 70 can be in the form of circular micro-holes, the diameter of which is between 0.1 micron and 10 microns, and the distance between the center points of two adjacent micro-holes is between 0.1 micron and 20 microns.
  • the diameter of the micro-holes can also be corresponding The size of the hole and the size of the center point of the microhole.
  • the long side of the transparent substrate 10 ranges from 10-100 mm, and the short side ranges from 5-50 mm.
  • the length, width and height of the transparent base 10 can be 75mm*25mm*1mm, and of course the size of the transparent base 10 can be designed accordingly according to actual needs.
  • the microporous structure 70 includes a flared portion 71 on the surface of the transparent substrate 10 and a constricted portion 72 inside the transparent substrate 10.
  • the flared portion 71 communicate with the constriction portion 72 along the depth direction of the transparent substrate 10 to form a microporous structure 70 on the transparent substrate 10 .
  • the microporous structure 70 is a funnel-shaped deep cone structure in the depth direction, and the diameter of the flaring portion 71 formed on the surface of the transparent substrate 10 is larger than that of the constricted portion 72 formed inside the transparent substrate 10.
  • the diameter, that is, the diameter of the microporous structure 70 gradually decreases from the surface of the transparent substrate 10 to the inside.
  • the diameter of the flaring portion 71 is larger, which facilitates the positioning of the coded microspheres and makes them easier to place.
  • the inclination angle of the deep cone structure is between 0° and 60°, that is, the angle between the line connecting the flaring portion 71 and the constricting portion 72 and the depth direction of the transparent substrate 10 is between 0° and 60°.
  • this angle can be processed and adjusted according to actual needs.
  • the transparent substrate 10 has a first surface 11 and a second surface 12, and the first surface 11 and the second surface 12 are processed by photolithography and engraving respectively.
  • the microporous region 70 is formed by etching; wherein, the first surface 11 and the second surface 12 are two opposite surfaces of the transparent substrate 10 .
  • the second Other processes can be performed on the surface 12 (ie, the bottom surface), such as pre-treatment or post-treatment such as cleaning the micropore area, which can improve the overall work efficiency.
  • This preferred embodiment provides a size design of a spatial transcriptome biochip.
  • the length, width and thickness of the glass substrate are 75mm, 25mm and 1mm respectively, and eight rectangular sub-regions 30 are arranged on the glass substrate, and four are arranged along the length direction. Rows, two columns are set along the width direction, the distance between the rectangular sub-region 30 and the top of the glass substrate is 8mm, the distance from the side of the glass substrate is 3.3mm, the distance between the two columns of rectangular sub-regions 30 is 4mm, and the distance between the adjacent rows
  • the rectangular sub-areas 30 are 5 mm apart.
  • the length and width of each rectangular sub-region 30 are 7.2 mm and 7.2 mm, respectively.
  • the pore diameter of the microporous structure 70 etched on the transparent substrate 10 is about 2.5um, which is equivalent to increasing the resolution of the current mainstream spatial transcriptome by more than 20 times.
  • HE staining and gene expression experiments can also be performed on glass slides later.
  • the human body is composed of a variety of tissues, and the cells in the tissues are different in type, time and space, so it is particularly important to study spatial specificity.
  • Space omics can preserve the integrity of the spatial structure of the sample through tissue slices, so as to obtain the gene expression in different regions. Therefore, spatial analysis at the subcellular level is an important means to study the functions of tissues and organs and the vital activities of living organisms.
  • Biochips can be manufactured by synthesizing microspheres modified with different sequences. Each microsphere has its corresponding probe. The microspheres are mixed and embedded in the holes on the surface of the glass plate, and then the chip is decoded by different fluorescent probes. So as to obtain the decoding chip.
  • the decoded biochip can capture mRNA in tissue sections in situ and perform subsequent sequencing steps to obtain transcriptome sequence information with tissue spatial location information. This sequencing method can achieve multi-cell, single-cell and subcellular resolution Tissue-space transcriptome sequencing.
  • the traditional biochip one-step hybridization reaction obtains less information and has low decoding efficiency. Therefore, the development of a three-color fluorescence decoding method is conducive to better research on the spatial position information of tissues at the subcellular level.
  • a three-color fluorescence decoding method of a subcellular spatial chip is provided.
  • the biochip involved in this method is prepared by embedding silica microspheres modified with different sequences to be decoded into a glass substrate with holes on the surface, and then performing multiple hybridization reactions on the chip with probes labeled with different fluorescent dyes , so as to obtain the decoded biochip.
  • This method can also introduce more fluorescent dyes to achieve fewer decoding times.
  • Design primer sequences and probe sequences design coding primer sequences and corresponding decoding probe sequences with different fluorescent labels.
  • Probe hybridization reaction The decoded probes with different fluorescent labels in each round are mixed and then hybridized with the chip, and then the corresponding fluorescent signals are detected.
  • Chip melting and cleaning melt the hybridized chip with sodium hydroxide, and clean the chip for the next round of hybridization reaction.
  • Chip decoding The microsphere sequence is decoded according to each round of fluorescence signals of each well on the chip.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • FIG. 1 The rendering of the silicon dioxide chip of the present invention before assembly is shown in FIG. 1 .
  • FIG. 2 The rendering of the silicon dioxide chip of the present invention before assembly is shown in FIG. 2 , and the rendering of the silicon dioxide chip and microspheres after self-assembly is shown in FIG. 3 .
  • the chip was incubated on a 95°C metal bath module for 30 minutes, then washed three times with ultrapure water, scanned in a scanner, and compared with the quality control chart before thermal incubation. The results showed that high temperature treatment would not affect the structure of the chip. The porosity is still higher than 99%.
  • Embodiment 2 The optimization that ultraviolet glue selects
  • UV glue manufacturers Hole drop rate Extraordinary force 3217 shadowless glue 99% Kingstar UV shadowless glue 83% ergo1309 Swiss imported AB glue 50%
  • the silica microsphere solution was prepared with ultrapure water, and three microsphere spin-coating concentration gradients of 10W/ ⁇ l, 20W/ ⁇ l, and 30W/ ⁇ l were set respectively, and 20W/ ⁇ l was finally determined as the optimum spin-coating concentration.
  • microsphere resuspension buffer a. Ultrapure water; b. Three DMSO solutions with concentrations of 5%, 10%, and 20%; c. Concentrations of 2.5 ⁇ , 5 ⁇ , and 10 ⁇ UV-curable glue solution. Finally, the 5 ⁇ UV-curable glue solution was determined to be the most suitable microsphere resuspension buffer.
  • test buffers namely a, ultrapure water; b, DMSO solutions with concentrations of 5, 10, and 20% respectively; c, UV curing with concentrations of 2.5, 5, and 10% respectively Glue solution; after counting on a hemocytometer, re-dilute the microsphere concentration to 20W/ ⁇ l.
  • Microsphere Resuspension Buffer Hole drop rate Ultra-pure water 50% 5% DMSO solution 40% 10% DMSO solution 59% 20% DMSO solution 75% 2.5 ⁇ UV curable glue solution 89% 5 ⁇ UV curable glue solution 99% 10 ⁇ UV curable glue solution 93%
  • Adopting the method for preparing the microsphere chip of the present invention not only has a high dropout rate, but also the microspheres are not easy to fall off, which is stronger than the method of directly dropping holes through physical extrusion.
  • Embodiment 5 The method for synthesizing Barcode silica microspheres by connection method
  • This example provides a method for synthesizing silica microspheres encoded by a long sequence by the ligation method, using silica microsphere microcarriers to connect the microspheres to the first oligonucleotide sequence through a condensation reaction, and then linking the microspheres through T4
  • the enzyme continues to extend the subsequent second and third primers, and finally obtains microspheres encoded by various long sequences.
  • the method comprises the steps of:
  • the above-mentioned activated silica microspheres were evenly divided into 96-well plates, 20 ⁇ L per well, and then 5 ⁇ L of amino-modified oligonucleotides (primer 1 dissolved in 0.1 M MES, with a final concentration of 60 ⁇ M) were added to each well. ), oscillating and mixing, setting the vibration speed of the metal oscillator to 300-2000rpm, and oscillating and reacting at room temperature overnight.
  • microspheres After the reaction, collect the microspheres into a 50mL centrifuge tube, wash once with PBS containing 0.001-0.03% v/v Tween-20, carefully remove the supernatant after centrifugation, and then wash the microspheres twice in TE Buffer, Resuspend the microspheres in enzyme-free water.
  • Primer 2 and primer 3 were mixed at a molar ratio of 1:1, and then 5 ⁇ annealing buffer was added for annealing reaction; the PCR program was set as: 95°C-15°C, annealing at 10°C every 3 minutes, and finally Barcode 2 primers (annealed products) were obtained. Primer concentration 100 ⁇ M.
  • the 5 ⁇ annealing buffer formula is as follows:
  • Double distilled water Make up to a total volume of 1000 ⁇ L
  • Barcode 3 ligation reaction distribute the resuspended silica microspheres in step 4 to a 96-well plate, then add 5 ⁇ L T4 ligation buffer, 500 U/ ⁇ L T4 ligase 5 ⁇ L, 100 ⁇ M Barcode 3 primer (primer 4) 5 ⁇ L, the remaining water, the total volume is 50 ⁇ L. React on a metal bath shaker at 16°C for 0.5-3h (vibration speed 300-2000rpm).
  • the primers in Table 4 involve Linker 1 and Linker 2 of different lengths, but are not limited to the length combinations shown above, and the length of the linker is 1-20nt.
  • the quality inspection probe sequence used is: 5′-cy5-AAAAAAAAAAAAAAAA-3′ (SEQ ID NO: 25)
  • the fluorescent quality inspection picture of the silica microspheres synthesized by the ligation method of the present invention with long sequence coding is shown in FIG. 6 .
  • the synthesis method of the long-sequence-coded silica microspheres provided in this example comprises the following steps:
  • EDC and NHS Accurately weigh 1.09mg EDC and 0.65mg NHS, prepare 0.1M MES at the same time, dissolve the weighed EDC and NHS with 100ul MES, and obtain a mixture of EDC and NHS; ), washed twice with the prepared MES solution, and then the above-mentioned EDC and NHS mixture was added to the microspheres, with a final reaction volume of 100 ⁇ L; then the microspheres were placed at room temperature, and the metal bath was shaken (2000rpm) for 30 minutes.
  • microspheres After the microspheres were reacted, they were evenly divided into 5 tubes, and then 2.5 ⁇ L of amino-modified oligonucleotides (dissolved in 0.1M MES, with a final concentration of 50 ⁇ M) were added to each tube, mixed by pipetting, and then kept at room temperature. Next, the metal bath was shaken (2000rpm) to react overnight. After the reaction was completed, collect the microspheres into 1 mL of 0.1 M PBS containing 0.02% Tween 20, centrifuge, carefully remove the supernatant, and then wash the microspheres twice in 1 ml TE buffer (pH 8.0).
  • amino-modified oligonucleotides dissolved in 0.1M MES, with a final concentration of 50 ⁇ M
  • connection steps of barcode 3 are the same as those of barcode 2, and the oligonucleotide sequences added are: reverse complementary sequence to linker 3, a unique barcode reverse complementary sequence and linker 2 reverse complementary sequence; after barcode 3 connection cleaning is completed Then add 2 ⁇ L of UMI for the same treatment, where the added UMI sequence is: a poly A tail, UMI sequence and reverse complementary sequence of Linker 3. Repeat the barcode 2 connection step.
  • Microspheres were stored in TE-TW solution (10mM Tris pH 8.0; 1mM EDTA, 0.01% Tween 20) at 4°C.
  • N and B are degenerate bases, N stands for A, T, C or G, and B stands for G, T or C.
  • Example 8 Three-color fluorescence decoding technology based on subcellular level biochip
  • primer 1 barcode1
  • primer 2 barcode2
  • primer 3 barcode3
  • each set of primers included 384 sequences.
  • the primer 1 sequence is first connected to the carboxyl silica microsphere through the aminocarboxyl condensation reaction. After the connection is completed, the microsphere is mixed and cleaned, and evenly divided into 384 parts. Each part is added with the primer 2 sequence for the ligation reaction. Repeat the operation until the primer 3 sequence is connected. Spread the prepared microspheres evenly on a microporous glass plate after mixing, and fix them at the hole positions to obtain a space chip.
  • the prepared chip needs to go through multiple rounds of decoding before finally determining the sequence structure of each hole microsphere.
  • the decoding probe is an oligonucleotide single-stranded structure complementary to the primer 1, primer 2 and primer 3 carried by the microsphere, and the decoding probe labeled with a fluorescent dye is selected from DAPI, FITC, Alexa fluor 488, CY2, Cy3, Three fluorescent dyes were selected from Cy5, CY5.5, TRITC, Cy7 and other fluorescent dyes, and probes with unlabeled fluorescent dyes were defined as dark. Three fluorescent dyes mark the three sections of Barcode 1, 2, and 3, respectively.
  • each round of hybridization solution for each well is 50-200 ⁇ L in total, including 40-190 ⁇ L of hybridization buffer (1-10mM NaCl, 2-5mM Tris-HCl, 1-3mM MgCl2 and 0.5-5mM DTT), 10 ⁇ L (1nM-50nM) three color mixed probes.
  • hybridization buffer (1-10mM NaCl, 2-5mM Tris-HCl, 1-3mM MgCl2 and 0.5-5mM DTT
  • 10 ⁇ L 1nM-50nM
  • chip fluorescence collection After chip fluorescence collection, take 100 ⁇ L 0.1-2M NaOH and place it on the well for 1-10 minutes to ensure complete melting. Repeat 2-3 times, and then wash with NFW 1-3 times to ensure that the hybridization probe is cleaned to avoid affecting Next round of reactions. After the chip is washed and dried, the next round of decoding probes is hybridized again, and the operation is repeated to complete 2-9 rounds of decoding processes.
  • Table 6 Fluorescently labeled probes for each decoding cycle.
  • Table 7 is the combination of fluorescent signals corresponding to each primer sequence.
  • Barcode decoding method Bar1-1 Fluorescence 1 - Fluorescence 1 Bar1-2 Fluorescent 1-Black Bar1-3 Black-Fluorescent 1 Bar1-4 black-black Bar2-1 Fluorescence 2 - Fluorescence 2 Bar2-2 Fluorescent 2-Black Bar2-3 Black-Fluorescent 2 Bar2-4 black-black Bar3-1 Fluorescence 3 - Fluorescence 3 Bar3-2 Fluorescent 3-Black Bar3-3 Black-Fluorescent 3 Bar3-4 black-black
  • Table 8 shows the sequences of primers 1, 2, and 3, and the sequence of the decoding probe (the reverse complementary sequence of the barcode).
  • the decoding probe needs to be labeled Mix it later.
  • V represents A, G or C
  • N represents A, T, G or C

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Abstract

L'invention concerne un procédé de préparation d'une puce à microsphères et une application de celle-ci. Le procédé de préparation de la puce à microsphères comprend : l'utilisation d'une lame de verre en dioxyde de silicium gravée avec des micropores comme substrat, l'application uniforme d'une couche d'adhésif ultraviolet sur la lame de verre en dioxyde de silicium, puis l'ajout de microsphères de dioxyde de silicium codées, et la centrifugation pour obtenir une puce biologique possédant une capacité de décodage spatial.
PCT/CN2022/140097 2021-12-21 2022-12-19 Procédé de préparation d'une puce à microsphères et application associée WO2023116639A1 (fr)

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CN202210137482.XA CN114410764B (zh) 2022-02-15 2022-02-15 长dna序列二氧化硅微珠的制备方法
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CN202220960413.4U CN216712064U (zh) 2022-04-25 2022-04-25 空间转录组芯片
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CN202211139855.3A CN115725700A (zh) 2022-09-19 2022-09-19 基于亚细胞水平空间芯片的三色荧光解码方法
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CN114350745A (zh) * 2021-12-21 2022-04-15 北京百迈客生物科技有限公司 微球芯片的制备方法
CN114410764A (zh) * 2022-02-15 2022-04-29 北京百迈客生物科技有限公司 长dna序列二氧化硅微珠的制备方法
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CN105925572A (zh) * 2016-06-07 2016-09-07 厦门大学 一种dna编码微球及其合成方法
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CN117089599B (zh) * 2023-10-20 2024-02-13 青岛百创智能制造技术有限公司 一种长编码序列微珠及其制备方法

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